Physiology Metabolism Characteristic And Related Genes Identification Of Mulberry In Response To Boron Stress

Boron is an essential micronutrient for normal growth and development of plants.It plays an important role in cell wall formation,cell membrane homeostasis,pollen tube growth,carbohydrate metabolism and photosynthesis.Given the narrow range between boron deficiency and excess,plants are vulnerable to the adverse effects of boron stress.Mulberry is an eco-economic crop with the homology of medicine and food,and the cultivation of excellent and highly resistant mulberry varieties is of great significance to the development of the sericulture industry,ecological management and medicinal fields.In this experiment,the effect of different concentrations of low boron and high boron treatment on physiological and biochemical indicators,photosynthetic characteristics,metabolome and transcriptome of mulberry were analyzed,and the mulberry xyloglucan endotransglucosylase/hydrolase MmXTH23 gene was silenced by virus induced gene silencing（VIGS）technology to identify its function.The response mechanism of mulberry to boron stress was revealed from physiological,metabolic and molecular levels,in order to provide a theoretical basis for cultivating new mulberry varieties with efficient utilization of boron.The main results are as follows:1.Analysis of physiological and biochemical indicators and photosynthetic characteristics of mulberry under boron stressUnder low and high boron stress,the stomatal conductance of mulberry leaves decreased,and the chlorophyll（Chl）content decreased.It was speculated that boron stress hinder the photosynthesis of mulberry.The accumulation of soluble protein,soluble sugar and proline（PRO）contributed to the maintenance of osmotic balance.The increase of malondialdehyde（MDA）content indicated the aggravation of membrane lipid peroxidation.The decrease of superoxide dismutase（SOD）and catalase（CAT）activity indicated that antioxidant enzyme systems were compromised.The increase of peroxidase activity（POD）may be beneficial to alleviate oxidative damage.The decrease of net photosynthetic rate（Pn）,transpiration rate（Tr）,stomatal conductance（Gs）,and intercellular CO₂ concentration（Ci）indicated that boron stress inhibited photosynthetic characteristics of mulberry.2.Metabolomic analysis of mulberry in response to boron stressA total of 1781 metabolites were detected in mulberry leaves by liquid chromatograph mass spectrometer（LC-MS）technology,including carboxylic acids and derivatives,fatty acyls,organooxygen compounds,prenol lipids,benzene and substituted derivatives,flavonoids,steroids and derivatives.A total of 108,134,89 and 110 differential metabolites（DMs）were identified in the 0,0.02,0.5 and 1 m M H₃BO₃ treatment groups,respectively.Alterations of amino acids,secondary metabolites,carbohydrates and lipids may be important material basis for mulberry to response to boron stress.KEGG pathway enrichment analysis revealed that DMs were mainly enriched in amino acid metabolism,biosynthesis of other secondary metabolites,lipid metabolism,metabolism of cofactors and vitamins and metabolism of other amino acids.3.Transcriptome analysis of mulberry in response to boron stressA total of 28,460 genes were detected in mulberry leaves by transcriptome sequencing technology,including 27,085 known genes and 1375 novel predicted genes.A total of 1734,721,1319 and 2340 differentially expressed genes（DEGs）were identified in the 0,0.02,0.5and 1 m M H₃BO₃ treatment groups,respectively,of which 206 were common in all four groups.Sixteen DEGs were randomly selected from the transcriptome data for quantitative real-time PCR（qPCR）detection to verify the accuracy of transcriptome sequencing results.KEGG pathway enrichment analysis revealed that the DEGs were mainly enriched in flavonoid biosynthesis,MAPK signaling pathway-plant and plant-pathogen interaction,suggesting that these pathways play an important role in the response of mulberry to boron stress.4.Cloning and functional identification of mulberry MmXTH23 geneThe MmXTH23 gene was cloned from mulberry.The length of CDS region sequence was 870 bp,encoding 289 amino acids,belonging to the glycoside hydrolase GH16 family.The protein molecular weight was 32211.93,the theoretical isoelectric point was 5.28,and there was a transmembrane domain at the N-terminus.The protein instability coefficient was31.29,which was predicted to be a stable protein.The average hydrophobic coefficient of the protein was-0.347,which was predicted to be a hydrophilic protein.The results of qPCR indicated that both low and high boron treatments up-regulated the expression level of MmXTH23 gene.The pTRV2-GFP-MmXTH23 recombinant vector was constructed by VIGS technology,and the MmXTH23 gene was successfully silenced.The relative expression levels of MmXTH23 gene were detected on days 12,14,16 and 18 after infestation,respectively.The results indicated that the gene was most efficiently silenced on day 16 after infestation.The pectin content in mulberry leaves was determined on day 16 after infection.The results showed that the total pectin content of mulberry leaves did not change significantly,while the water-soluble pectin content increased and the chelator-soluble pectin content decreased in the infestation group.It is hypothesized that the MmXTH23 gene may affect the cross-linking state of pectin in cell wall by altering the proportion of different types of pectin content in mulberry leaves,thus playing a role in response of mulberry to boron stress.